Prior to the present invention, proteins such as antibodies have been radiolabeled with iodine. A more attractive approach to the radiolabeling of amines, polypeptide chains and proteins has been the "bifunctional chelate" methods in which a strong chelating group is covalently bonded to the protein which, in turn, is labeled with a variety of chelatable radionuclides. In the resultant product, the protein retains its biological function and the product also retains the radionuclide. When the chelating group chosen forms stable chelates, the radiolabel is likely to be stable in vivo. In addition, its presence will have only a minor effect on the specificity or other functional biochemical characteristics of the protein.
Although methods have been developed for labeling uncoupled proteins with .sup.99m Tc, both the mechanism of labeling and the site of attachment are unknown. More importantly, the labeled protein is often contaminated with loosely bound or unbound chemical forms of .sup.99m Tc. To minimize these radiocontaminants, various labeling methods have exposed the protein to acidic conditions, employed long incubation periods and/or required terminal chromatography for purification. In addition to increasing the rate of blood clearance and uptake in several organs, these radiocontaminants may permit exchange of .sup.99m Tc among plasma proteins.
It has also been proposed to label proteins linked to DTPA with .sup.99m Tc in the presence of dithionite reducing agent, [Khaw et al, Nuclear Med., vol. 23, 00. 1011-1019 (1982)]. However, it has not been shown that the DTPA groups bound to the protein are labeled with .sup.99m Tc. Although these methods differ in important respects, they all rely upon the protein to stabilize .sup.99m Tc in a reduced oxidation state and prevent both re-oxidation and the formation of radiocolloids. Proteins may be only partially successful in this judging by in vivo instabilities reported in the prior art: when the biological behavior of .sup.99m Tc-labeled proteins have been compared to radioiodinated proteins, with very few exceptions, .sup.99m Tc was found to clear more rapidly from blood. Other investigations have reported increased liver accumulation.
Since DTPA-coupled proteins may be stabily labeled with .sup.111 In, the use of DTPA-coupled proteins for .sup.99m Tc labeling could offer the advantage of greater in vivo stability than that presently attainable. However, investigations into the labeling of coupled proteins with .sup.99m Tc, it has been observed that proteins can compete with DTPA for .sup.99m Tc [Lanteigne et al, J. Nuc. Med., 24:23 (1983) Abstract].
Overall, therefore, there is a long demonstrated need for a simple and efficient means for utilizing a bifunctional chelating agent for binding radiolabels to amines, polypeptides and proteins and other medically important compounds such that their radiolabeled forms are stable in vivo and retain their specific biological activities to a substantial degree.